Svetlana I. Galkina

Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co‐cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria‐resembling structures were identified. Moreover, after co‐cultivation with cardiomyocytes, expression of human‐specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture

Cell-to-cell interactions of human mesenchymal multipotent stromal cells (MMSC) and rat renal tubular cells (RTC) were explored under conditions of co-cultivation. We observed formation of different types of intercellular contacts, including so called tunneling nanotubes. These contacts were shown to be able to provide transfer of cells contents, including organelles. We documented intercellular exchange with fluorescent probes specific to cytosol, plasmalemma and mitochondria. Initial transport of cellular components was revealed after 3 h of co-culturing, and occurred in two directions--both direct and retrograde as referred to RTC. However, transport of probes toward MMSC was more efficient. One significant result of such transport was appearance of renal-specific Tamm-Horsfall protein in MMSC, indicating induction of their differentiation into kidney tubular cells. We conclude that transfer of cellular compartments between renal and stem cells could provide differentiation of MMSC when transplanted into kidney and result in therapeutic benefits in renal failure.

Protective effect of mitochondria-targeted antioxidants in an acute bacterial infection

Significance The main approach to treat acute pyelonephritis is antibiotic therapy. However, the pathology is accompanied by inflammation and oxidative stress phenomena that can also be a target for intervention when direct antibacterial measures are impossible or inefficient. In our study, in vitro and in vivo models of experimental pyelonephritis were used to define the role of mitochondria in this pathology and to find a way to alleviate the kidney damage. The majority of the deleterious effects of pyelonephritis, including animal mortality in extreme cases, were prevented by the treatment with the mitochondria-targeted antioxidant, pointing to mitochondria as a therapeutic target. Acute pyelonephritis is a potentially life-threatening infection of the upper urinary tract. Inflammatory response and the accompanying oxidative stress can contribute to kidney tissue damage, resulting in infection-induced intoxication that can become fatal in the absence of antibiotic therapy. Here, we show that pyelonephritis was associated with oxidative stress and renal cell death. Oxidative stress observed in pyelonephritic kidney was accompanied by a reduced level of mitochondrial B-cell lymphoma 2 (Bcl-2). Importantly, renal cell death and animal mortality were both alleviated by mitochondria-targeted antioxidant 10(6′-plastoquinonyl) decylrhodamine 19 (SkQR1). These findings suggest that pyelonephritis can be treated by reducing mitochondrial reactive oxygen species and thus by protecting mitochondrial integrity and lowering kidney damage.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) beta 2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Involvement of ecto‐ATPase and extracellular ATP in polymorphonuclear granulocyte‐endothelial interactions

We conclude that PMN‐endothelial adhesion is counteracted by an ecto‐ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Cell-cell contacts alter intracellular pH

Intracellular pH, an important regulatory factor for many cellular activities, was shown to be modulated by cell adhesion to the solid substratum. In the present work we have shown that cell-cell contacts also affect intracellular pH. pH(i) depends on how many contacts the cell has established with the substratum and the neighboring cells. pH(i) is low in single cells, not contacting each other. It increased with the increase of cell density. pH(i) is again decreased in confluent (topoinhibited) monolayers. pH(i)-shifts triggered by cell-cell contacts seem to be mediated by Na+/H(+)-antiporter. Dependence of pH(i) on cell density could be simulated by different concentration of Arg-Gly-Asp--which is part of the site of extracellular matrix proteins involved in integrin binding. The dependence of pH(i) on cell-cell contacts is discussed in relation to the phenomena of topoinhibition.

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes, membrane tethers) in human neutrophils

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm. The extensions were filled with cytoplasm and covered with membrane, as they included fluorescent cytoplasmic and lipid dyes. Neither protein kinase C inhibitors H-7 and bisindolylmaleimide VII, nor tyrosine protein kinase inhibitors tyrphostin AG 82 and genistein caused such extensions formation. Supposedly, STS induces membrane tubular extension formation promoting actin cytoskeleton depolymerization or affecting NO synthesis.

Nitric oxide mediates distinct effects of various LPS chemotypes on phagocytosis and leukotriene synthesis in human neutrophils

We investigated the effect of lipopolysaccharide (LPS) chemotypes differing in their carbohydrate chain length on phagocytosis of serum-opsonized zymosan (OZ) particles and related functions of human polymorphonuclear leukocyte (PMNL, neutrophils). LPS from deep core mutant (Re), complete core (Ra) and smooth (S) phenotypes of Salmonella typhimurium was studied. Priming of neutrophils with various LPSs caused prominent enhancement of OZ phagocytosis, superoxide production and leukotriene (LT) synthesis in neutrophils, with LPS effects increasing as Re<S<Ra. The LPS forms were less potent to activate OZ uptake in the presence of MK-886, 5-lipoxygenase activating protein inhibitor, suggesting the regulatory function of 5-lipoxygenase (5-LO)-derived LTs. Direct measurement of nitrite release from OZ-stimulated neutrophils revealed that the effects of LPS on NO synthesis increased in the range of Ra<S<Re. Nitric oxide synthase (NOS) inhibitor l-NAME increased phagocytosis, LT and superoxide formation by neutrophils, and abolished the difference in the action of the LPSs forms. Further mechanistic studies revealed that NO modulates cellular 5-LO activity in a guanylyl cyclase and protein kinase G dependent manner, as well as interplay between NO and superoxide, and peroxynitrite generation contribute to distinct effects of LPS chemotypes on phagocytosis and LT synthesis in human neutrophils. Our investigation of the three LPS species demonstrates that the LPS polysaccharide core is mostly essential for the PMNL activation and is able to suppress lipid A-induced increase in NOS activity in phagocyting cells by triggering specific signaling cascades.

Membrane tubules attach Salmonella Typhimurium to eukaryotic cells and bacteria

Using scanning electron microscopy techniques we measured the diameter of adhesive tubular appendages of Salmonella enterica serovar S. Typhimurium. The appendages interconnected bacteria in biofilms grown on gallstones or coverslips, or attached bacteria to host cells (human neutrophils). The tubular appendage diameter of bacteria of virulent flagellated C53 strain varied between 60 and 70 nm, thus considerably exceeding in size of flagella or pili. Nonflagellated bacteria of mutant SJW 880 strain in biofilms grown on gallstones or coverslips were also interconnected by 60-90-nm tubular appendages. Transmission electron microscopy studies of thin sections of S. Typhimurium biofilms grown on agar or coverslips revealed numerous fragments of membrane tubular and vesicular structures between bacteria of both flagellated and nonflagellated strains. The membrane structures had the same diameter as tubular appendages observed by scanning electron microscopy, indicating that tubular appendages might represent membrane tubules (tethers). Previously, we have shown that neutrophils can contact cells and bacteria over distance via membrane tubulovesicular extensions (TVE) (cytonemes). The present electron microscopy study revealed the similarities in size and behavior of bacterial tubular appendages and neutrophil TVE. Our data support the hypothesis that bacteria establish long-range adhesive interactions via membrane tubules.

Lipid-cell interactions. Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites.

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and 31P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.